Wnt/β‐catenin signaling is hyperactivated in systemic sclerosis and induces Smad‐dependent fibrotic responses in mesenchymal cells

Objective

Fibrosis in human diseases and animal models is associated with aberrant Wnt/β‐catenin pathway activation. The aim of this study was to characterize the regulation, activity, mechanism of action, and significance of Wnt/β‐catenin signaling in the context of systemic sclerosis (SSc).

Methods

The expression of Wnt signaling pathway components in SSc skin biopsy specimens was analyzed. The regulation of profibrotic responses by canonical Wnt/β‐catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied using proliferation, migration, and gel contraction assays. The cell fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.

Results

Analysis of published genome‐wide expression data revealed elevated expression of the Wnt receptor FZD2 and the Wnt target LEF1 and decreased expression of Wnt antagonists DKK2 and WIF1 in skin biopsy specimens from subsets of patients with diffuse cutaneous SSc compared to the other distinct subsets. Immunohistochemical analysis showed increased nuclear β‐catenin expression in these biopsy specimens. In vitro, Wnt‐3a induced β‐catenin activation, stimulated fibroblast proliferation and migration, collagen gel contraction, and myofibroblast differentiation, and enhanced profibrotic gene expression. Genetic and pharmacologic approaches were used to demonstrate that these profibrotic responses involved autocrine transforming growth factor β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes, Wnt‐3a repressed adipogenesis and promoted myofibroblast differentiation.

Conclusion

Canonical Wnt signaling was hyperactivated in SSc skin biopsy specimens. In explanted mesenchymal cells, Wnt‐3a stimulated fibrogenic responses while suppressing adipogenesis. Taken together, these results indicate that Wnts have potent profibrotic effects, and that canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

     

Stimulation of superficial zone protein accumulation by hedgehog and Wnt signaling in surface zone bovine articular chondrocytes

Objective

To determine the roles of the hedgehog and Wnt signaling pathways in accumulation of superficial zone protein (SZP) in surface zone articular chondrocytes.

Methods

Explant cultures of disks of surface zone cartilage or isolated chondrocytes from the surface zone of articular cartilage of bovine stifle joints were cultured in serum‐free chemically defined medium. Accumulation of SZP in the culture medium, in response to hedgehog proteins (sonic hedgehog [SHH] and Indian hedgehog [IHH]), Wnt proteins (Wnt‐3a, Wnt‐5a, and Wnt‐11), agonists of the Wnt/β‐catenin pathway (glycogen synthase kinase 3β [GSK‐3β] inhibitors), and antagonists of the Wnt/β‐catenin pathway, was investigated. The interaction between transforming growth factor β1 (TGFβ1) and hedgehog proteins or antagonists of the Wnt/β‐catenin pathway was also investigated.

Results

Hedgehog proteins stimulated SZP accumulation. Activation of the Wnt/β‐catenin pathway by Wnt‐3a and GSK‐3β inhibitors led to inhibition of SZP accumulation; however, Wnt‐5a and Wnt‐11 had no influence on SZP accumulation. Conversely, antagonists of the Wnt/β‐catenin pathway stimulated SZP accumulation. In addition, there were combinatorial effects of TGFβ1 and hedgehog proteins or antagonists of the Wnt/β‐catenin pathway on SZP accumulation.

Conclusion

SHH and IHH signaling has a stimulatory effect on SZP accumulation in surface zone cartilage and isolated articular chondrocytes. These findings provide insight into the regulatory mechanisms of articular cartilage homeostasis and maintenance by morphogens.

     

Effectiveness of anti–folate receptor β antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages

Objective

To define the distribution of folate receptor β (FRβ)–expressing cells in various tissues, including rheumatoid arthritis (RA) synovial tissues, and to verify the effects of an immunotoxin composed of an anti‐FRβ monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA) on apoptosis and tumor necrosis factor α (TNFα) production by adherent synovial mononuclear cells from RA patients.

Methods

Anti‐FRβ mAb were produced by immunizing mice with FRβ‐transfected murine pre–B cells. The distribution of the FRβ antigen was examined by immunohistochemical analysis using anti‐FRβ mAb and macrophage‐specific anti‐CD163 mAb. Anti‐FRβ mAb was chemically crosslinked with truncated PEA. FRβ‐expressing macrophages were produced by the transfection of adenovirus vector containing the FRβ gene. Apoptotic cells were detected by staining with propidium iodide. TNFα was measured by enzyme‐linked immunosorbent assay.

Results

FRβ‐expressing cells were not present in peripheral blood leukocytes and their activated cells. In all of the tissues examined, most FRβ‐expressing cells were CD163+. The immunotoxin significantly induced the apoptosis of FRβ‐transfected macrophages and adherent RA synovial mononuclear cells and inhibited TNFα production by adherent RA synovial mononuclear cells.

Conclusion

We demonstrated the limited distribution of FRβ‐expressing cells in various tissues. The immunotoxin targeting FRβ‐expressing cells will provide a therapeutic tool for rheumatoid synovitis.

     

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

Objective

Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA.

Methods

Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/β‐catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression.

Results

Wnt‐induced signaling protein 1 (WISP‐1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt‐16 and Wnt‐2B were also markedly up‐regulated during the course of disease. Interestingly, increased WISP‐1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP‐1 resulted in interleukin‐1–independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP‐1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage.

Conclusion

This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP‐1 expression is a feature of experimental and human OA and that WISP‐1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

     

Interleukin‐1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt‐5a/receptor tyrosine kinase–like orphan receptor 2 pathway

Objective

Mesenchymal stem cells (MSCs) are considered to be a novel tool for the treatment of rheumatoid arthritis (RA) because of their multipotency to differentiate into osteoblasts and chondrocytes, their immunosuppressive effects, and availability. The aim of this study was to assess the mechanisms of human MSC differentiation into osteoblasts under inflammatory conditions.

Methods

Human MSCs were cultured in commercialized osteogenic induction medium with inflammatory cytokines for up to 10 days. Osteoblast differentiation was detected by alkaline phosphatase staining and messenger RNA (mRNA) expression of multiple osteoblast markers. Mineralization was assessed by alizarin red S staining.

Results

Among the various cytokines tested, interleukin‐1β (IL‐1β) induced differentiation of human MSCs into osteoblasts, which was confirmed by alkaline phosphatase activity, expression of RUNX2 mRNA, and strong alizarin red S staining. Among various molecules of the Wnt family, Wnt‐5a and receptor tyrosine kinase–like orphan receptor 2 (Ror2), a major receptor of Wnt‐5a, were significantly induced in human MSCs by IL‐1β. Silencing of either WNT5A or ROR2 by small interfering RNA with 2 different sequences reduced alkaline phosphatase activity, RUNX2 expression, and alizarin red S staining of human MSCs induced by IL‐1β.

Conclusion

IL‐1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt‐5a/Ror2 pathway. These results suggest potential benefits of IL‐1β–treated human MSCs in the treatment of damaged bone as well as in the induction of self‐renewal and self‐repair of damaged tissue, including osseous tissue.

     

Estrone/17β‐estradiol conversion to,and tumor necrosis factor inhibition by,estrogen metabolites in synovial cells of patients with rheumatoid arthritis and patients with osteoarthritis

Objective

The role of estrogens in rheumatoid arthritis (RA) is debated since both proinflammatory and antiinflammatory effects have been reported. Important evidence of the dual role of estrogens is conversion to various proinflammatory or antiinflammatory metabolites. This study was undertaken to examine the downstream conversion of estrogens in synovial cells from patients with RA or osteoarthritis (OA).

Methods

We studied serum levels of estrone, estrone sulfate, and estrone sulfate membrane transporters, intracellular interconversion of estrone and 17β‐estradiol, and conversion of estrone/17β‐estradiol to various estrogen metabolites in RA and OA synovial cells. The effect of estrogen metabolites on tumor necrosis factor (TNF) secretion was also studied in RA and OA synovial cells.

Results

Serum levels of estrone sulfate were similar in healthy controls and RA patients. Estrone sulfate transporters were present in synovial tissue. Interconversion of estrone and 17β‐estradiol and the expression of converting enzymes of the cytochrome P450 family were similar in RA and OA cells. Using estrone and 17β‐estradiol as substrates, RA and OA synovial cells produced 16α‐, 4‐, and 2‐hydroxylated estrogens and their 4‐ and 2‐methylation products. The levels of 16α‐hydroxylated estrone/17β‐estradiol (16αOH‐estrone/16αOH‐17β‐estradiol) were higher than the levels of all other estrogen metabolites. RA synovial cells produced more 16αOH‐estrone than did OA synovial cells. Importantly, the 16αOH estrogens did not inhibit TNF secretion, whereas all other estrogen metabolites had marked inhibitory effects.

Conclusion

Our findings indicate that precursor estrogens are converted to proinflammatory metabolites, particularly in RA synovial cells. RA synovial cells mainly produce the proproliferative 16αOH‐estrone, which, in addition to 16αOH‐17β‐estradiol, is one of the only 2 estrogens studied that does not inhibit TNF secretion. A preponderance of 16α‐hydroxylated estrogens is an unfavorable sign in synovial inflammation.

     

In vitro and in vivo efficacy of a recombinant immunotoxin against folate receptor β on the activation and proliferation of rheumatoid arthritis synovial cells

Objective

To investigate the effects of the recombinant immunotoxin dsFv anti‐FRβ‐PE38, which consists of the disulfide‐stabilized Fv fragment (dsFv) of the anti–folate receptor β (anti‐FRβ) antibody and the 38‐kd portion of Pseudomonas exotoxin A (PE38), on the activation and proliferation of cells that function in inflammatory and degradative processes in rheumatoid arthritis (RA) synovial tissue.

Methods

The Ig V_H‐PE38 fusion protein and the Ig V_L protein were produced in Escherichia coli, and then joined with a disulfide bond by engineering cysteine residues in the framework regions of these proteins. The effects of dsFv anti‐FRβ‐PE38 on the activation and proliferation of cells in RA synovial tissue were investigated by immunohistochemistry; the numbers of cells expressing CD68, vascular cell adhesion molecule 1, angiopoietin 1, CD34, proliferating cell nuclear antigen, and interleukin‐6 and the numbers of apoptotic cells were counted in RA synovial tissue engrafted into SCID mice treated or not treated with dsFv anti‐FRβ‐PE38. The effects of dsFv anti‐FRβ‐PE38 on the generation of osteoclasts from RA adherent synovial mononuclear cells in vitro was investigated by counting the number of resorption pits on dentin slices treated or not treated with dsFv anti‐FRβ‐PE38.

Results

Administration of dsFv anti‐FRβ‐PE38 reduced the numbers of macrophages, activated fibroblast‐like cells, endothelial cells, and proliferating cells and increased the numbers of apoptotic cells in RA synovial tissue engrafted into SCID mice. In vitro, the generation of osteoclasts from RA adherent synovial mononuclear cells was largely suppressed by treatment with dsFv anti‐FRβ‐PE38.

Conclusion

Our findings show that dsFv anti‐FRβ‐PE38 immunotoxin would be a promising tool for the treatment of RA synovitis, especially when administered intraarticularly.

     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

Effect of intermittent administration of human parathyroid hormone on bone mineral density and arthritis in rats with collagen‐induced arthritis

Objective

To investigate the effect of intermittent administration of human parathyroid hormone (PTH) on bone mineral density (BMD) and arthritis in rats with collagen‐induced arthritis (CIA).

Methods

Seven‐month‐old female Sprague‐Dawley rats were divided into 4 groups: rats without CIA (control), rats with CIA treated with vehicle, rats with CIA treated with PTH for 4 weeks, and rats with CIA treated with PTH for 6 weeks. PTH (20 μg/kg) was injected subcutaneously 3 times per week. BMD in the proximal metaphysis and the diaphysis of the tibia was measured by peripheral quantitative computed tomography every 2 weeks until week 8. Eight weeks after initial sensitization, the animals were killed, and the BMD and mechanical properties of excised limbs were evaluated. Histomorphometric analysis of tibiae and histologic evaluation of arthritis were also performed.

Results

In the PTH‐treated rats with CIA, the incidence and severity of arthritis were macroscopically and histologically similar to the findings in the vehicle‐treated rats with CIA. The decrease of BMD caused by CIA was suppressed by treatment with human PTH, in a manner that was dependent on the duration of administration. In the histomorphometric analysis, bone formation parameters were higher and bone resorption parameters were lower in the PTH‐treated arthritic rats compared with vehicle‐treated arthritic rats. Mechanical properties were also maintained in the PTH‐treated rats.

Conclusion

Our findings indicate that, in an animal model of arthritis, intermittent PTH administration activates bone formation, resulting in increased BMD and preventing deterioration of mechanical properties. However, PTH has no effect on the arthritis itself.

     

Rosiglitazone induces interleukin‐1 receptor antagonist in interleukin‐1β–stimulated rat synovial fibroblasts via a peroxisome proliferator–activated receptor β/δ–dependent mechanism

Objective

To study the potency of 2 peroxisome proliferator–activated receptor γ (PPARγ) agonists, 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐deoxy‐PGJ₂) and rosiglitazone, to modulate the expression of interleukin‐1 receptor antagonist (IL‐1Ra) in rat synovial fibroblasts.

Methods

Levels of messenger RNA for IL‐1Ra and PPAR isotypes (α, β/δ, γ) were assessed by real‐time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL‐1β. PPAR levels were assessed by Western blotting and secreted IL‐1Ra levels by immunoassay. The potency of PPARγ agonists and the PPARβ/δ agonist GW‐501516 on IL‐1Ra levels was tested in the range of 1–10 μM and at 100 pM, respectively. The contribution of PPARγ to the effects of rosiglitazone on IL‐1Ra secretion was examined either by its overexpression or by inhibition using wild‐type or dominant‐negative constructs and the antagonist GW‐9662 (10 μM), respectively. The dominant‐negative strategy was also performed to investigate the possible contribution of PPARβ/δ and NF‐κB activation.

Results

IL‐1β–induced IL‐1Ra production was increased by 10 μM rosiglitazone but was reduced dose‐dependently by 15‐deoxy‐PGJ₂. Both agonists lowered IL‐1β secretion, but rosiglitazone alone reduced the imbalance of IL‐1β/IL‐1Ra toward basal levels. Enhancement of IL‐1β–induced IL‐1Ra production by rosiglitazone was not affected by PPARγ overexpression or by its inhibition with dominant‐negative PPARγ or GW‐9662. Inhibition of NF‐κB was also ineffective against rosiglitazone but abolished the stimulating effect of IL‐1β on IL‐1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARγ decreased dramatically upon IL‐1β exposure, whereas PPARβ/δ remained stable. Dominant‐negative PPARβ/δ abolished the enhancement of IL‐1Ra by rosiglitazone, whereas GW‐501516 reproduced the effect of rosiglitazone on IL‐1Ra secretion.

Conclusion

Rosiglitazone stimulates IL‐1Ra production by a PPARβ/δ mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.

     

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin‐1β

Objective

Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin‐1β (IL‐1β), and combinations thereof.

Methods

Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17β‐estradiol, and dehydroepiandrosterone (DHEA), with or without IL‐1β. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix.

Results

Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 μg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration‐dependent stimulation of integration (with maximum values observed with DHEA at 3 × 10⁻⁵M, testosterone at 10⁻⁸M, and 17β‐estradiol at 10⁻¹¹M). For testosterone and 17β‐estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL‐1β at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL‐1β and sex hormones, integration of articular cartilage was reduced.

Conclusion

This is the first study to demonstrate that steroid hormones such as 17β‐estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL‐1β. In the absence of IL‐1β or after neutralization of IL‐1β, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.